The Laboratory Chief has acted as principle investigator for a $3.8 million collaborative contract with DARPA to determine if a diet of ketone esters can enhance the metabolic and cognitive performance of war fighters under conditions of extreme physiological and emotional stress. The project involves: a) the production of esters of D-?O-hydroxybutyrate obtained the product of bacterial fermentation poly D-?O-hydroxybutyrate in collaboration with John Hammerstone of Mars Inc, b) determine the physiological and cognitive effects in collaboration with Prof. Kieran Clarke of Oxford University, c) test the ketone ester diet for toxicity in rodents and rodent newborns in collaboration with Prof. Carl Keen, UC Davis d) construct a non-invasive optical monitoring system to determine the level of ketosis in animals and man in collaboration with Prof Britton Chance, U of Pennsylvania e) determine, in the Laboratory of Metabolic Control, NIAAA, the levels and duration of ketosis achieved on feeding the ketone ester diet and the changes in intermediary and energy metabolites in heart and brain, and in collaboration with Drs George Kunos and Dr Douglas Osei-hyiaman of the Laboratory of Physiological Studies, NIAAA, changes in important neuropeptides that bear on changes in cognitive function during ketosis. f) model in silico, the metabolic changes that would occur during ketosis in collaboration with Dr. David Pollidori, Entelos Corporation. The projects outlined above are scheduled for completion by June, 2005. Assuming the goals of this project are met, then application will be made to DARPA for funds to formulate and test the effects of a ketone ester diet on the physiological and cognitive performance in military recruits undergoing different forms of extreme training exercises. The theoretical justification on which more efficient physiological performance during mild ketosis is anticipated have been described previously (1) where we showed that cardiac hydraulic efficiency could be increased by about 28%. The role of ketosis in overcoming insulin resistance, which accompanies extreme physical and psychological stress have also been described (2). Classically, ketosis has been induced by feeding high fat, low carbohydrate diets, however such diets can induce significant elevation in blood lipids with atherogenic potential (3). Alternative high fat diets have been proposed utilizing mid chain or unsaturated fatty acids (4;5), but are not well tolerated by patients. More importantly the feeding of high fat diets inevitably leads to the elevation of blood free fatty acids with consequent activation of the PPAR nuclear transcription system increasing the transcription of uncoupling proteins leading to metabolic inefficiency with decreased cardiac phosphorylation potential which can induce cardiac arrythmias and in some cases failure or unstable angina. The alternative to the feeding of a high fat diet is to create ketone esters which are hydrolysable by gut into ketone bodies. A ready and cheap source of poly D-?O-hydroxybutyrate is available as a fermentation product from Alcaligenes eutrophus (6). While the bacterial polymer in its native form is not digestible in gut, short esters are. The process to create such esters have involved new and novel means of production which are patentable. In addition to the military uses of such compounds, it appears that mild ketosis would be of therapeutic benefit in 3 major disease phenotypes: 1) diseases of substrate deficiency, such as Alzheimer's disease, types I & II diabetes, and insulin resistant states, 2) diseases of free radical toxicity, such as Parkinson's disease, amyotrophic lateral sclerosis and reperfusion injury and 3) disease of hypoxia, such as myocardial infarction and stroke. The medical uses of such compounds are also being investigated by Dr. Yoshihiro Kashiwaya, MD PhD in a visiting scientist in our laboratory. Duchenne?s and Becker?s muscular dystrophy is the most common monogenetic disease affecting 1/3,600 male births. The genetic lesion is a failure to synthesis dystrophin an structural muscle protein. Attempts to express dystrophin or eutrophin in these patients by molecular biological means have so far failed. Accordingly, there is no treatment for these patients with the result that they are wheelchair bound by the end of the first decade of life and die in the 2nd decade of either heart or respiratory failure. It has been found, by Prof Kieran Clarke and her collegues at Oxford University (25), that the mouse model of Duchenne?s , the mdx mouse, has impaired glucose uptake, possibly due to a decreased translocation of GLUT4 in response to insulin stimulation. This raises the possibility that these patients could be treated by inducing mild ketosis (2). We have therefore developed a system to measure the hydraulic efficiency in the working perfused mouse heart, a technique not accomplished by any other laboratory and one requiring considerable technical skill. We have now shown that the decreased hydraulic efficiency of working perfused MDX mouse hearts can be improved by addition of 5 mM Na D-?-hydroxybutyrate to the perfusion media. This raises the possibility of a potentially life saving form of therapy for these patients, for which no therapy now exists. During last year, and in collaboration with Dr. Roscoe Brady of NINDS we prepared applications in response to a DoD solicitation for new therapeutic approaches in the areas of 1) epilepsy, 2) Duchenne?s muscular dystrophy, and 3) amyotrophic lateral sclerosis. Unfortunately these applications were not successful and no funds were made available. However, I believe the approach outlines was sound, and if our present program of ester synthesis with DARPA is successful, these applications and the approach outlined should be submitted to NIH for further consideration. This material has been the subject of 2 meetings sponsored by the NIH Office of Rare Disease over the past 2 years. Dr Kashiwaya in collaboration with Dr. Ray Masuda, a Fogarty Fellow has also examined the effects of ketone bodies on hypoxia in cultured hippocampal neurons. A manuscript reporting the results of this work is in preparation. Significance to the Programs of the NIAAA Furtherance of defenses against bioterrorism is one of the priorities in the NIH roadmap and this collaboration with the Department of Defense is consonant with this NIH priority. The possibility of developing a new therapy for a presently untreatable diseases is of significance to the mission of the NIH and this institute.